U.S. patent application number 10/553695 was filed with the patent office on 2006-11-30 for composition for nkt cell activation.
This patent application is currently assigned to KIBUN FOOD CHEMIFA CO., LTD.. Invention is credited to Kazuyoshi Kawahara, Yoshio Kumazawa, Katsumi Murata, Hiroaki Takimoto.
Application Number | 20060269524 10/553695 |
Document ID | / |
Family ID | 34879308 |
Filed Date | 2006-11-30 |
United States Patent
Application |
20060269524 |
Kind Code |
A1 |
Kumazawa; Yoshio ; et
al. |
November 30, 2006 |
Composition for nkt cell activation
Abstract
Provided is more effective a cell activator. A cell activator
comprising a glycosphingolipid having a structure represented by
the following formula (1): formula (1) ##STR1## wherein R.sup.1
represents the following formula (1-1): formula (1-1) ##STR2##
wherein R.sup.3 represents alkyl or alkenyl and R.sup.4 represents
alkyl; and R.sup.2 represents hydrogen, or .alpha.-galactose,
.alpha.-glucose, .alpha.-mannose, .alpha.-glucosamine,
.beta.-glucosamine or a combination thereof.
Inventors: |
Kumazawa; Yoshio; (Kanagawa,
JP) ; Murata; Katsumi; (Tokyo, JP) ; Kawahara;
Kazuyoshi; (Tokyo, JP) ; Takimoto; Hiroaki;
(Tokyo, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
KIBUN FOOD CHEMIFA CO.,
LTD.
Tokyo
JP
Yoshi Kumazawa
Kawasaki-shi
JP
|
Family ID: |
34879308 |
Appl. No.: |
10/553695 |
Filed: |
February 21, 2005 |
PCT Filed: |
February 21, 2005 |
PCT NO: |
PCT/JP05/03234 |
371 Date: |
August 7, 2006 |
Current U.S.
Class: |
424/93.7 ;
514/54 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 31/7012 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/093.7 ;
514/054 |
International
Class: |
A61K 35/14 20060101
A61K035/14; A61K 31/739 20060101 A61K031/739 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 19, 2004 |
JP |
2004-043481 |
Claims
1-26. (canceled)
27. A cell activator comprising a glycosphingolipid having a
structure represented by the following formula (1) ##STR22##
wherein R.sup.1 represents the following formula (1-1): ##STR23##
wherein R.sup.3 represents alkyl or alkenyl and R.sup.4 represents
alkyl; and R.sup.2 represents hydrogen, or .alpha.-galactose,
.alpha.-glucose, .alpha.-mannose, .alpha.-glucosamine,
.beta.-glucosamine or a combination thereof.
28. A cell activator comprising a glycosphingolipid having a
structure represented by the following formula (3): ##STR24##
wherein R.sup.5 represents R.sup.51, R.sup.52, R.sup.53, R.sup.54,
R.sup.55, R.sup.56, R.sup.57, R.sup.58, R.sup.59, R.sup.70,
R.sup.71, R.sup.72, R.sup.73, R.sup.74, R.sup.75, R.sup.76,
R.sup.77, or R.sup.78; and R.sup.6 represents hydrogen, R.sup.62,
R.sup.63, R.sup.64, or R.sup.65: ##STR25## ##STR26## ##STR27##
##STR28## ##STR29## ##STR30## ##STR31## ##STR32## ##STR33##
##STR34##
29. A method of activating NKT cell which comprises administering
the cell activator according to claim 27 to a mammal.
30. A method of activating NKT cell which comprises administering
the cell activator according to claim 28 to a mammal,
31. A method of accelerating IL-4 production which comprises
administering the cell activator according to claim 27 to a
mammal.
32. A method of accelerating IL-4 production which comprises
administering the cell activator according to claim 28 to a
mammal.
33. A method of accelerating IFN-.gamma. production which comprises
administering the cell activator according to claim 27 to a
mammal.
34. A method of accelerating IFN-.gamma. production which comprises
administering the cell activator according to claim 28 to a
mammal.
35. A method of activating dendritic cell which comprises
administering the cell activator according to claim 27 to a
mammal.
36. A method of activating dendritic cell which comprises
administering the cell activator to claim 28 to a mammal.
37. A method of accelerating IL-12 production which comprises
administering the cell activator according to claim 27 to a
mammal.
38. A method of accelerating IL-12 production which comprises
administering the cell activator according to claim 28 to a
mammal.
39. A method of accelerating IL-10 production which comprises
administering the cell activator according to claim 27 to a
mammal.
40. A method of accelerating IL-10 production which comprises
administering the cell activator according to claim 28 to a
mammal.
41. A method of activating NK cell which comprises administering
the cell activator according to claim 27 to a mammal.
42. A method of activating NK cell which comprises administering
the cell activator according to claim 28 to a mammal.
43. A method for treatment or prophylaxis of tumor comprises
administering the cell activator according to claim 27 to a
mammal.
44. A method for treatment or prophylaxis of tumor comprises
administering the cell activator according to claim 28 to a
mammal.
45. A method for treatment or prophylaxis of allergy comprises
administering the cell activator according to claim 27 to a
mammal.
46. A method for treatment or prophylaxis of allergy comprises
administering the cell activator according to claim 28 to a
mammal.
47. A method of enhancing resistance to infection which comprises
administering the cell activator according to claim 27 to a
mammal.
48. A method of enhancing resistance to infection which comprises
administering the cell activator according to claim 28 to a
mammal.
49. A method of inhibiting viral activity which comprises
administering the cell according to claim 27 to a mammal.
50. A method of inhibiting viral activity which comprises
administering the cell activator according to claim 28 to a
mammal.
51. A method of accelerating IL-6 production which comprises
administering the cell activator according to claim 27 to a
mammal.
52. A method of accelerating IL-6 production which comprises
administering the cell activator according to claim 28 to a
mammal.
53. A method of accelerating NO production which comprises
administering the cell activator according to claim 27 to a
mammal.
54. A method of accelerating NO production which comprises
administering the cell activator according to claim 28 to a mammal.
Description
[0001] The present application is a continuation of
PCT/JP2005/003234 filed on Feb. 21, 2005 and claims priority under
35 U.S.C. .sctn.119 of Japanese Patent Application No. 043481/2004
filed on Feb. 19, 2004.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a composition for NKT cell
activation, a composition for accelerating IL-4 production, a
composition for accelerating IFN-.gamma. production, a composition
for dendritic cell activation, a composition for accelerating IL-12
production, a composition for accelerating IL-10 production, a
composition for NK cell activation, an antitumor composition, an
antiallergic composition, a composition for enhancing resistance to
infection, an antiviral composition, a composition for accelerating
IL-6 production, and a composition for accelerating NO production,
comprising a glycosphingolipid with a given structure.
[0004] 2. Description of the Related Art
[0005] It has been heretofore considered that glycosphingolipids
are present on the surface layers of animal cells or the like and
that they are associated with the recognition mechanism. In
contrast, gram-negative bacteria have outer membranes consisting of
lipopolysaccharides, proteins, and phosphoric acids, on their cell
cortexes, and they interact with the outer world via the outer
membranes. Accordingly, lipopolysaccharides as the primary
components of the outer membranes had been considered to be present
in and to be essential for all gram-negative bacteria. In recent
years, however, it has become known that the aerobic gram-negative
bacteria Sphingomonas paucimobilis, previously known as
"Pseudomonas paucimobilis," do not comprise lipopolysaccharides and
comprise glycosphingolipids as bacterial lipids.
[0006] The present inventors have succeeded in isolating the
glycolipids from the aforementioned Sphingomonas paucimobilis,
analyzing the chemical structure thereof, and identifying the same
(WO 92/12986). Also, the present inventors have disclosed that the
aforementioned glycosphingolipids have excellent moisturizing
effects and barrier effects and thus are extensively applicable as
cosmetics (JP Patent Publication No. 11-43437). Further, the
present inventors have elucidated the fact that the aforementioned
glycosphingolipids have excellent emulsifying effects (JP Patent
Publication No. 2000-51676).
[0007] The present inventors have also disclosed that
glycosphingolipids obtained from bacterial strains of the other
genus Sphingomonas are excellent as cosmetic and pharmaceutical
compositions (JP Patent Publication No. 2002-010797).
[0008] It has been reported that the NKT cells expressing T cell
receptors (TCR) are related to NK cells in the following respect:
they exhibit large granular lymphocyte-like (LGL-like) in
morphologies, they constantly express the IL-2R .beta. chain, and
they have perfolin. However, they are completely different from NK
cells in terms of the possession of TCR (J. Immunol., 155, 2972,
1995).
[0009] Given the circumstances, it has been reported that mouse NKT
cells that express NK1.1 in IL-12-activated T cells are important
effector cells for inhibiting hematogenous metastasis of tumors to
the liver or lung (J. Immunol., 154, 4333, 1995 and J. Immunol.,
88, 82, 1996).
[0010] As described above, the NKT cells have drawn attention as a
new group of cells in recent years.
[0011] Further, WO 98/44928 describes that .alpha.-glycosylceramide
having a given structure is effective as an NKT cell activator. A
more effective NKT cell activator has been desired.
SUMMARY OF THE INVENTION
[0012] An object of the present invention is to overcome the
aforementioned problems and provide more effective for cell
activator.
[0013] More specially, the object of the present invention is to
provide a composition for NKT cell activation, a composition for
accelerating IL-4 production, a composition for accelerating
IFN-.gamma. production, a composition for dendritic cell
activation, a composition for accelerating IL-12 production, a
composition for accelerating IL-10 production, a composition for NK
cell activation, an antitumor composition, an antiallergic
composition, a composition for enhancing resistance to infection,
an antiviral composition, a composition for accelerating IL-6
production, and a composition for accelerating NO production,
comprising glycosphingolipid.
[0014] Under the above circumstances, the present inventors found
that the object could be attained by the following means.
[0015] (1) A composition for NK cell activation comprising a
glycosphingolipid having a structure represented by the following
formula (1): ##STR3## wherein R.sup.1 represents the following
formula (1-1): formula (1-1) ##STR4## wherein R.sup.3 represents
alkyl or alkenyl and R.sup.4 represents alkyl; and
[0016] R.sup.2 represents hydrogen, or .alpha.-galactose,
.alpha.-glucose, .alpha.-mannose, .alpha.-glucosamine,
.beta.-glucosamine or a combination thereof.
[0017] (2) The composition for NKT cell activation according to
(1), wherein the formula (1) is represented by the following
formula (3): ##STR5## wherein R.sup.5 represents R.sup.51,
R.sup.52, R.sup.53, R.sup.54, R.sup.55, R.sup.56, R.sup.57,
R.sup.58, R.sup.59, R.sup.70, R.sup.71, R.sup.72, R.sup.73,
R.sup.74, R.sup.75, R.sup.76, R.sup.77, or R.sup.78; and R.sup.6
represents hydrogen, R.sup.62, R.sup.63, R.sup.64, or R.sup.65:
##STR6## ##STR7## ##STR8## ##STR9## ##STR10## ##STR11## ##STR12##
##STR13## ##STR14## ##STR15##
[0018] (3) A composition for accelerating IL-4 production
comprising a glycosphingolipid having a structure represented by
the following formula (1).
[0019] (4) The composition for accelerating IL-4 production
according to (3), wherein the formula (1) is represented by the
following formula (3).
[0020] (5) A composition for accelerating IFN-.gamma. production
comprising a glycosphingolipid having a structure represented by
the following formula (1).
[0021] (6) The composition for accelerating IFN-.gamma. production
according to (5), wherein the formula (1) is represented by the
following formula (3).
[0022] (7) A composition for dendritic cell activation comprising a
glycosphingolipid having a structure represented by the following
formula (1).
[0023] (8) The composition for dendritic cell activation according
to (7), wherein the formula (1) is represented by the following
formula (3).
[0024] (9) A composition for accelerating IL-12 production
comprising a glycosphingolipid having a structure represented by
the following formula (1).
[0025] (10) The composition for accelerating IL-12 production
according to (9), wherein the formula (1) is represented by the
following formula (3).
[0026] (11) A composition for accelerating IL-10 production
comprising a glycosphingolipid having a structure represented by
the following formula (1).
[0027] (12) The composition for accelerating IL-10 production
according to (11), wherein the formula (1) is represented by the
following formula (3).
[0028] (13) A composition for NK cell activation comprising a
glycosphingolipid having a structure represented by the following
formula (1).
[0029] (14) The composition for NK cell activation according to
(13), wherein the formula (1) is represented by the following
formula (3).
[0030] (15) An antitumor composition comprising a glycosphingolipid
having a structure represented by the following formula (1).
[0031] (16) The antitumor composition according to (15), wherein
the formula (1) is represented by the following formula (3).
[0032] (17) An antiallergic composition comprising a
glycosphingolipid having a structure represented by the following
formula (1).
[0033] (18) The antiallergic composition according to (17), wherein
the formula (1) is represented by the following formula (3).
[0034] (19) A composition for enhancing resistance to infection
comprising a glycosphingolipid having a structure represented by
the following formula (1).
[0035] (20) The composition for enhancing resistance to infection
according to (19), wherein the formula (1) is represented by the
following formula (3).
[0036] (21) An antiviral composition comprising a glycosphingolipid
having a structure represented by the following formula (1).
[0037] (22) The antiviral composition according to (21), wherein
the formula (1) is represented by the following formula (3).
[0038] (23) A composition for accelerating IL-6 production
comprising a glycosphingolipid having a structure represented by
the following formula (1).
[0039] (24) The composition for accelerating IL-6 production
according to (23), wherein the formula (1) is represented by the
following formula (3).
[0040] (25) A composition for accelerating NO production comprising
a glycosphingolipid having a structure represented by the following
formula (1).
[0041] (26) The composition for accelerating NO production
according to (25), wherein the formula (1) is represented by the
following formula (3).
[0042] Further, the present invention provides a method of
activating NKT cell, a method of accelerating IL-4 production, a
method of accelerating IFN-.gamma. production, a method of
activating dendritic cell, a method of accelerating IL-12
production, a method of accelerating IL-10 production, a method of
activating NK cell, a method for treatment or prophylaxis of tumor,
a method for treatment or prophylaxis of allergy, a method of
enhancing resistance to infection, a method of inhibiting viral
activity, a method of accelerating IL-6 production, and a method of
accelerating NO production by in which these methods comprise
administering the cell activator to a mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1 shows the analysis results of flow cytometry when
GSL-1 has been administered to a normal mouse.
[0044] FIG. 2 shows the analysis results of flow cytometry when
GSL-2 has been administered to a normal mouse.
[0045] FIG. 3 shows the analysis results of flow cytometry when
GSL-6 has been administered to a normal mouse.
[0046] FIG. 4 shows the analysis results of flow cytometry when
GSL-7 has been administered to a normal mouse.
[0047] FIG. 5 shows the analysis results of flow cytometry when
GSL-1 has been administered to a TLR4-deficient mouse.
[0048] FIG. 6 shows the analysis results of flow cytometry when
GSL-2 has been administered to a TLR4-deficient mouse.
[0049] FIG. 7 shows NKT cell changes when GSL-1, 2, 6, or 7 has
been administered to a normal mouse.
[0050] FIG. 8 shows IFN-.gamma. concentration after GSL-1 or GSL-2
has been administered to a normal mouse.
[0051] FIG. 9 shows changes in IFN-.gamma.-producing NKT cells when
GSL-1 or GSL-2 has been administered to a normal mouse.
[0052] FIG. 10 shows IL-4 concentration after GSL-1 or GSL-2 has
been administered to a TLR4-deficient mouse.
BEST MODE OF CARRYING OUT THE INVENTION
[0053] Hereafter, the present invention is described in detail. In
the present specification, the symbol ".about." is used to denote
the numerical values that precede or follow the same as lower
limits or upper limits. In this specification, R.sup.51, R.sup.52,
R.sup.53, R.sup.54, R.sup.55, R.sup.56, R.sup.57, R.sup.58,
R.sup.59, R.sup.70, R.sup.71, R.sup.72, R.sup.73, R.sup.74,
R.sup.75, R.sup.76, R.sup.77, and R.sup.78 in formula (3) may thus
be represented as "R.sup.51.about.R.sup.78."
[0054] The glycosphingolipid that is used in the present invention
(hereafter they may be referred to as "GSL(s)") have the structure
represented by the formula (1). Alkyl represented by R.sup.3 that
is included in R.sup.1 in formula (1) may contain cycloalkyl, and
the cycloalkyl may be present at the alkyl terminus or in the alkyl
chain. A preferable example of cycloalkyl is cyclopropane. Alkyl
represented by R.sup.3 has preferably 13 to 23, and more preferably
15, 16, 17, 18, 19, 20 or 21, carbon atoms. Alkyl or alkenyl
represented by R.sup.3 preferably has a substituted or
unsubstituted straight chain. A double bond may be present at any
position in alkenyl.
[0055] In contrast, alkyl represented by R.sup.4 has preferably 10
to 20 carbon atoms, more preferably 10, 11, 12, 13, 14, 15 or 16
carbon atoms, and further preferably 10, 11, 12, 13, 14 or 15
carbon atoms. Alkyl represented by R.sup.4 preferably has a
substituted or unsubstituted straight chain.
[0056] More preferably, R.sup.1 is any of the aforementioned
R.sup.51.about.R.sup.78.
[0057] R.sup.1 in formula (1) preferably represents the
conformation represented by the following formula (1-2): ##STR16##
wherein R.sup.3 and R.sup.4 are as defined above in formula (1-1).
Thus, the aforementioned R.sup.51.about.R.sup.78 having such
conformations are more preferable.
[0058] Formula (1) is preferably represented by the formula (2),
(3), (4), or (5). Formula (2) is as shown below: ##STR17## wherein
R.sup.1 and R.sup.2 are as defined above in formula (1), and their
preferable ranges are also as defined above.
[0059] In a preferable structure represented by the formula (3),
R.sup.5 is any of R.sup.51.about.R.sup.78 and R.sup.6 is hydrogen
(hereafter the same may be referred to as "Structure A"), R.sup.5
is any of R.sup.51.about.R.sup.78 and R.sup.6 is R.sup.62
(hereafter the same may be referred to as "Structure B"), R.sup.5
is any of R.sup.51.about.R.sup.78 and R.sup.6 is R.sup.63
(hereafter the same may be referred to as "Structure C"), R.sup.5
is any of R.sup.51.about.R.sup.78 and R.sup.6 is R.sup.64
(hereafter the same may be referred to as "Structure D"), or
R.sup.5 is any of R.sup.51.about.R.sup.78 and R.sup.6 is R.sup.65
(hereafter the same may be referred to as "Structure E"). Further,
Structure A in which R.sup.5 is R.sup.51, R.sup.52, or R.sup.53 is
more preferably used.
[0060] Formula (4) is as shown below: ##STR18## wherein R.sup.1 is
as defined above in formula (1), and its preferable range is also
as defined above (a composition comprising at least one compound
represented by the formula (4) may be hereafter referred to as
"Structure AA").
[0061] Formula (5) is as shown below: ##STR19## wherein R.sup.1 is
as defined above in formula (1) (hereafter the same may be referred
to as "Structure F"), and its preferable range is also as defined
above.
[0062] In the present invention, a single type or two or more types
of glycosphingolipids may be used. When two or more types of
glycosphingolipids are used in combination, the proportion of each
component is not particularly limited. For example, a composition
comprising at least one of the 3 types of compounds having
Structure A, a composition comprising at least one of the 3 types
of compounds having Structure B, or a composition comprising at
least one of the compounds having Structure F may be used. Among
them, a structure in which R.sup.1 is any of R.sup.51, R.sup.52, or
R.sup.53 is preferable (hereafter the same may be referred to as
"Structure FA").
[0063] In addition to the composition described above, the
composition disclosed in the present invention may include
composition represented by the formula (1) wherein formula (1-1) is
represented by the conformation represented by the formula (1-1-1):
##STR20## wherein R.sup.3 and R.sup.4 are as defined above in
formula (1-1), and their preferable ranges are also as defined
above.
[0064] Formula (1-1-1) further preferably has the structure
represented by the formula (1-1-2): ##STR21## wherein R.sup.3 and
R.sup.4 are as defined above in the formula (1-1), and their
preferable ranges are also as defined above.
[0065] The glycosphingolipid represented by the formula (1) can be
extracted from bacteria having glycosphingolipid. For example,
methods disclosed in WO 92/12986 or JP Patent Publication No.
2002-10797 can be adopted. Since glycosphingolipid is contained in
bacteria of the genus Sphingomonas, the glycosphingolipid
represented by the formula (1) can be extracted from any of the
bacteria of the genus Sphingomonas. The bacteria of the genus
Sphingomonas include those that have been generally said to belong
to the genus Sphingomonas and those that are classified as
belonging to substantially the same genus as the genus
Sphingomonas. For example, any bacteria described in Microbiol.
Immunol., 2000, 44, 563-575 can be used in the present
invention.
[0066] The glycosphingolipids represented by the formula (1) are
insoluble in acetone. Accordingly, bacteria are preferably washed
with acetone before extraction. An alcoholic solvent such as
methanol or a mixed solvent comprising an alcoholic solvent mixed
with a polar solvent such as chloroform is preferably used for
extraction of the glycosphingolipid represented by the formula (1)
from the viewpoint of yield. Other types of solvents may be used as
long as such solvents are capable of dissolving
glycosphingolipid.
[0067] When a mixture of glycosphingolipids is obtained, components
thereof can be separated from one another in accordance with a
technique known in the art. For example, glycosphingolipids can be
completely separated via chromatography. When a mixed solution of
chloroform and methanol is used as an eluate, glycosphingolipids of
Structure A, Structure F, Structure C, and Structure B or Structure
D or Structure E, are eluted in that order. Since Structure B,
Structure D, and Structure E are generally produced by different
bacteria, glycosphingolipids can be very easily separated.
Conditions for separation via chromatography, such as a filler, an
eluate, a rate of elution, pressure, and temperature, can be
adequately regulated. Alternatively, a reagent that selectively
reacts with a specific substance contained in the glycosphingolipid
mixtures may be used in order to prepare a derivative of such
substance, and separation can be carried out with the utilization
of chemical or physical properties of such derivative. When the
bacterium Sphingomonas paucimobilis is used, glycosphingolipids of
Structure A and of Structure B are generally obtained. When the
bacterium Sphingomonas capsulata (nomen novum: Novosphingobium
capsulatum) is used, glycosphingolipids of Structure A and of
Structure C are generally obtained. When the bacterium Sphingomonas
adhaesiva is used, glycosphingolipids of Structure A and of
Structure D are generally obtained. When Sphingomonas sp. MK346 is
used, glycosphingolipids of Structure A and of Structure E are
generally obtained. When Sphingomonas wittichii, Sphingomonas
macrogoltabidus (nomen novum: Sphingopyxis macrogoltabida),
Sphingomonas terrae, or Sphingomonas yanoikuyae (nomen novum:
Sphingobium yanoikuyae) is used, glycosphingolipids of Structure AA
(e.g., Structure A) and of Structure F (e.g., Structure FA) are
generally obtained. Accordingly, glycosphingolipids of interest can
be efficiently obtained by selecting bacteria based on such
information.
[0068] The glycosphingolipid represented by the formula (1) can be
also synthesized by combining conventional synthesis techniques.
For example, a sugar and a sphingosine portion are first
synthesized or extracted from the bacteria. Then, the
glycosphingolipid represented by the formula (1) can be prepared by
forming amide bonds between the sugar and the sphingosine
portion.
[0069] In the present invention, NKT cells may include, for
example, human V.alpha.24.sup.+ NKT cells and mouse
V.alpha.14.sup.+ NKT cells. NKT cell activation may include
enhancement of cytotoxic activity, enhancement of cytokine
production, and acceleration of NKT cell proliferation. Further,
the composition for NKT cell activation of the present invention
accelerates the production of IL-4 and IFN-.gamma. as a
consequence. Accordingly, the composition disclosed in the present
invention can be used as accelerators of various functions
accelerated by IL-4 or IFN-.gamma.. Among the GSLs that are used in
the present invention, glycosphingolipid having galacturonic acid
are particularly preferable as the composition for NKT cell
activation.
[0070] Examples of functions accelerated by IL-4 may include Th2
induction and induction of antibody class switch. Examples of
functions accelerated by IFN-.gamma. may include Th1 induction and
macrophage activation.
[0071] In addition to the aforementioned, the compositions for
accelerating production or activation of each type of IL,
IFN-.alpha., IFN-.beta., tumor necrosis factors (TNF), lymphotoxin,
hematopoietic colony-stimulating factors (CSF), erythropoietin,
hematopoietic epidermal growth factors (EGF), and fibroblast growth
factors (FGF) can be used as enhancement of cytokine. Further, the
glycosphingolipids disclosed in the present invention can also be
used as the other immune-activating composition, an
apoptosis-inducing composition for cancer cells, or a composition
for accelerating production or activation targeting acceleration of
NF-kappaB activation, I-kappaB degradation, p38 phosphorylation, or
Akt phosphorylation.
[0072] In the present invention, dendritic cell activation may
include, for example, enhancement of antigen-presenting
capacity.
[0073] GSLs according to the present invention accelerate
production of IL-12 and of IL-10. Accordingly, such GSLs can also
be used as a composition for accelerating various functions which
are accelerated by the production of IL-12 and of IL-10.
Glycosphingolipid containing glucuronic acids are particularly
preferably used as the composition for accelerating IL-12 and/or
IL-10 production of the present invention.
[0074] In the present invention, NK cell activation may include,
for example, damage to infected cells. Among GSLs that are used in
the present invention, monosaccharide type-glycosphingolipids are
particularly preferably used as the composition for NK cell
activation.
[0075] In the present invention, antitumor effects may include, for
example, activation of helper T cells and of killer T cells.
Examples of particularly preferable the antitumor composition among
the GSLs used in the present invention may include
tetrasaccharide-type and monosaccharide-type glycosphingolipids.
The composition of the present invention can also be used for
treatment or prophylaxis of tumor, for example, B16 melanoma
F10(B16F10) cells in mammal. The mammal includes a human and an
animal.
[0076] In the present invention, antiallergic effect may include,
for example, inhibition of eosinophilic infiltration, inhibition of
mast cell activity, inhibition of IgE-production, and inhibition of
mediator release.
[0077] The antiallergic composition of the present invention
generally few side effects, and thus, application thereof for
pollenosis, asthma bronchiale, endogenous eczema, or the like is
particularly useful. Accordingly, the composition of the present
invention can also be preferably used for treatment or prophylaxis
of such allergy in mammal.
[0078] In the present invention, enhancing resistance to infection
may include, for example, activation of helper T cells and of
killer T cells. Application of the composition for enhancing
resistance to infection according to the present invention for
salmonella infection and tuberculosis is particularly useful. Among
GSLs used in the present invention, tetrasaccharide
type-glycosphingolipid is particularly preferable as the
composition for enhancing resistance to infection.
[0079] In the present invention, antiviral effect refers to
inhibiting viral activity, for example, enhancement of type-I
interferon production, activation of helper T cells, and activation
of killer T cells. The antiviral composition according to the
present invention for Herpesviridae infections such as
cytomegalovirus infection or herpesvirus infection is particularly
useful.
[0080] When the composition of the present invention is used as
pharmaceutical preparations, quasi-drugs, or active ingredients
thereof, the composition can be preferably administered as a
pharmaceutical composition that can be produced by a method known
in the art. Examples of pharmaceutical composition may include
tablets, capsules, powders, subtle granules, granules, liquids, and
syrups. Such pharmaceutical compositions can be produced with the
addition of pharmacologically or pharmaceutically acceptable
additives. Examples of pharmacologically or pharmaceutically
acceptable additives include vehicles, disintegrators or
disintegration assistants, binders, lubricants, coating agents,
dyes, diluents, bases, solubilizers or solubilization assistants,
isotonizing agents, pH regulators, stabilizers, propellants, and
adhesives. The pharmaceutical composition may also comprise one or
more ingredients having other effects without departing from the
scope of this invention. The dose of the pharmaceutical
preparations according to the present invention is not particularly
limited, and it can be adequately determined in accordance with the
type of active ingredient or other conditions. Such dose can be
adequately increased or decreased in accordance with a variety of
general factors such as the body weight or age of the patient, the
type or symptom of the disease, or the route of administration. In
general, the pharmaceutical composition can be administered in
amounts of 0.001 mg to 100 mg, and preferably 0.01 mg to 10 mg, per
adult per day. The route of administration is not particularly
limited, and administration can be made intravenously via injection
or infusion or orally.
EXAMPLES
[0081] The present invention is hereafter described in greater
detail with reference to the examples. The materials, the amounts
used, the percentages, the procedures, the processes, and other
conditions described in the following examples can be adequately
altered without departing from the scope of this invention.
Accordingly, the technical scope of the present invention is not
limited to the examples.
[0082] A composition containing Structure A produced from
Sphingomonas paucimobilis (GSL-1), a composition containing
Structure B produced from Sphingomonas paucimobilis (GSL-2), a
composition containing Structure C produced from Sphingomonas
capsulata (GSL-3), a composition containing Structure D produced
from Sphingomonas adhaesiva (GSL-4), a composition containing
Structure E produced from Sphingomonas sp. MK346 (GSL-5), a
composition containing Structure AA produced from Sphingomonas
yanoikuyae (GSL-6), a composition containing Structure F produced
from Sphingomonas yanoikuyae (GSL-7), a composition containing
Structure AA produced from Sphingomonas wittichii (GSL-8), a
composition containing Structure F produced from Sphingomonas
wittichii (GSL-9), a composition containing Structure AA produced
from Sphingomonas macrogoltabidus (GSL-10), a composition
containing Structure F produced from Sphingomonas macrogoltabidus
(GSL-11), a composition containing Structure AA produced from
Sphingomonas terrae (GSL-12), and a composition containing
Structure F produced from Sphingomonas terrae (GSL-13) were used in
the examples. The GSLs were separated in accordance with the
methods disclosed in WO 92/12986 and in JP Patent Publication No.
2002-010797.
Example 1
Assay of Activity of NKT Cell Activation
Animals:
[0083] C57BL/10ScSn mice (hereafter referred to as "normal mice")
and C57BL/10ScCr mice (TLR4-deficient/IL-12RB2 chain mutant mice)
(hereafter referred to as "TLR4-deficient mice;" 7-week-old,
female, the Max-Planck Institute of Immunobiology, Freiburg,
Germany) were used as test animals. GSL-1 and GSL-2 activate
macrophages of normal mice via TLR4 and induce IL-12 production.
TLR4-deficient mice have mutation in the .beta.-chain of the IL-12
receptor and thus IL-12 does not act. IL-12 is a substance that
potently activates the immune system. With the use of the
TLR4-deficient mice of the present example, acceleration of NKT
cell activation by the glycosphingolipid that are used in the
present invention can be further clarified without the influence of
IL-12 activity.
Preparation of Samples:
[0084] The GSLs (10 .mu.g) was administered to normal mice and to
TLR4-deficient mice. Administration was carried out via caudal
veins. Then, the serum and the liver were extracted from the mice,
one of which had been administered physiological saline as a
control (the control), another of which is one day after the
administration (day 1), and the other of which is two days after
the administration (day 2).
Separation of Intrahepatic Leukocytes:
[0085] The sampled liver was crushed using 2 glass slides. A cell
suspension was centrifuged at 500 rpm for 1 minute. The obtained
supernatant was centrifuged at 1,200 rpm (300 g) for 5 minutes. The
resulting sediment was suspended in 30% Percoll (Pharmacia), and
the resulting suspension was superposed on 67.5% Percoll,
centrifugation was carried out at 20.degree. C. at 2,000 rpm (800
g) for 30 minutes, and leukocytes were accumulated at the boundary
of 30% Percoll and 67.5% Percoll and then collected. The obtained
cells were washed three times with the Hanks medium to obtain
intrahepatic leukocytes.
Flow Cytometry:
[0086] In order to block nonspecific binding of a fluorescently
labeled antibody through FcR, anti-FcyR (2.4G2) was used. The
PE-labeled anti-NK1.1 antibodies (PK136, Nippon Becton Dickinson
Co., Ltd.) and the biotin-labeled anti-TCRap antibodies (H57-597,
Nippon Becton Dickinson Co., Ltd.) were used. After the antibodies
were added to the cells, the reaction was allowed to proceed at
4.degree. C. in the dark for 30 minutes. The cells treated with the
biotin-labeled antibodies were allowed to react with
Cy-chrome-bound streptavidin (554062, Nippon Becton Dickinson Co.,
Ltd.) at 4.degree. C. in the dark for 30 minutes. Staining of the
aforementioned antibodies and Cy-chrome-bound streptavidin and
washing of cells were carried out with the use of 0.1%
NaN3-containing 1% serum albumin. The cells were stained and then
immobilized with 1% paraformaldehyde-containing phosphate buffered
saline (PBS(-)). Assay was carried out using an automated cell
sorter (Coulter Epics Elite ESP, Beckman Coulter). This procedure
was separately carried out using the FITC-labeled anti-CD11a
antibodies (M17/4, Nippon Becton Dickinson Co., Ltd.) instead of
the PE-labeled anti-NK1.1 antibodies.
[0087] The flow cytometry was carried out by the method using the
PE-labeled anti-NK1.1 antibodies (or CD11a). The results thereof
are shown in FIGS. 1 to 6. FIGS. 1 to 4 sequentially show the cases
where GSL-1, GSL-2, GSL-6, and GSL-7 were administered to normal
mice. FIGS. 5 and 6 sequentially show the cases where GSL-1 and
GSL-2 were administered to TLR4-deficient mice.
[0088] In the drawings, numerical values represent the percentages
(%) of cells wherein both NK1.1 (or CD11a) and TCR.alpha..beta.
were expressed. It should be noted that the cells wherein both
NK1.1 (or CD11a) and TCR.alpha..beta. were expressed are NKT
cells.
[0089] The flow cytometry for normal mice was carried out in the
same manner as described above by the method using the PE-labeled
anti-NK1.1 antibodies on a different day. The results thereof (the
percentages of NKT cells) are shown in FIG. 7. In the drawing,
numerical values represent the percentages (%) of cells wherein
both NK1.1 (or CD11a) and TCR.alpha..beta. were expressed, and the
reference marks represent the cells that were found to be
statistically significantly different from the control as a result
of the t-test.
Confirmation of the Presence of IFN-.gamma.:
[0090] The IFN-.gamma. content in the blood after GSL-1 or GSL-2
had been administered was examined. GSL-1 or GSL-2 was administered
to the normal mice and to the TLR4-deficient mice in the same
manner as described above, and the IFN-.gamma. content in the serum
was analyzed.
[0091] The IFN-.gamma. content was analyzed by the sandwich ELISA
method using the purified anti-IFN-.gamma. antibodies (R.sup.4-6A2,
Nippon Becton Dickinson Co., Ltd.) and the biotin-conjugated
anti-IFN-.gamma. antibodies (AN-18, detection antibodies, Nippon
Becton Dickinson Co., Ltd.) as a detectable antibody. After the
reaction using the biotin-conjugated antibodies, alkaline
phosphatase-labeled streptavidin (43-4822, Zymed) was conjugated
thereto, and the color was developed using paranitrophenyl
phosphate (N-4645, Sigma) as a substrate. The absorbance at 405 nm
and that at 540 nm as the control were measured using a microplate
reader (Model: 550, Nippon BioRad Laboratories). As a result,
IFN-.gamma. production was found to be accelerated in the normal
mice, as shown in FIG. 8. Even though GSL-1 or GSL-2 was
administered to the TLR4-deficient mice, IFN-.gamma. was not
detected because they did not react with IL-12.
Flow Cytometry of IFN-.gamma.-Producing NKT Cells:
[0092] The normal mice to which GSL-1 or GSL-2 had been
administered were subjected to flow cytometry in the same manner as
described above, cells were stained with the PE-labeled anti-NK1.1
antibodies, the biotin-labeled anti-TCR.alpha..beta. antibodies,
and the FITC-labeled anti-IFN-.gamma. antibodies (554410, Nippon
Becton Dickinson Co., Ltd.), and the percentage of
IFN-.gamma.-producing NKT cells was determined. The results are
shown in FIG. 9. In the drawing, numerical values represent the
percentages of IFN-.gamma.-producing NKT cells wherein both NK1.1
and TCR.alpha..beta. were expressed.
Measurement of IL-4 Content:
[0093] Whether or not the IL-4 content in the blood increased after
GSL-1 or GSL-2 had been administered was examined. GSL-1 or GSL-2
was administered to the normal mice and to the TLR4-deficient mice
in the same manner as described above, and the IL-4 content in the
serum was analyzed.
[0094] The IL-4 content was measured by ELISA using the BD Opti-EIA
mouse IL-4 set (555232, Nippon Becton Dickinson Co., Ltd.) in
accordance with the instruction manual. As a result, increase of
IL-4 was observed in every mouse. In particular, IL-4 production
was significantly accelerated in the TLR4-deficient mice, as shown
in FIG. 10.
[0095] When GSL-1 or GSL-2 was added, increases in the percentages
of cells wherein both NK1.1 and TCR.alpha..beta. had been expressed
were observed via the aforementioned flow cytometry. In the cells
with significant increase in such expression levels, production of
both IFN-.gamma. and IL-4 was confirmed. This indicates that GSL-1
or GSL-2 is effective for activation of NKT cells.
Example 2
Acceleration of IFN-.gamma. Production
[0096] The C57BL/6 mice were used as test mice. Each type of GSL
was dissolved in 20% pentanediol to a final concentration of 10
mg/ml, the resulting solution was then dissolved in a solution
diluted with physiological saline (hereafter the same may be
referred to as "P") or a solution containing 0.5% pentanediol, 0.5%
N-lauroylsarcosine, and 9.8% sucrose to a final concentration of 5
mg/ml, and the GSL was then treated with a solution diluted with
physiological saline (hereafter the same may be referred to as
"P+S"). GSL was administered to mice via the caudal veins (dose:
100 .mu.g). The solvent alone was administered to mice as the
control (control). A cell suspension was prepared from the liver
extracted 16 hours after the administration, and intrahepatic
leukocytes were obtained via specific gravity centrifugation using
45% Percoll and 67.5% Percoll. The leukocytes were treated with the
anti-Fc.gamma.R antibodies (2.4G2) and then treated with the
FITC-labeled anti-IFN-.gamma. antibodies, the PE-labeled anti-NK1.1
antibodies, and the biotin-labeled anti-TCR.alpha..beta.
antibodies. After the antibodies were added to the leukocytes, the
reaction was allowed to proceed at 4.degree. C. in the dark for 30
minutes. The leukocytes treated with the biotin-labeled antibodies
were allowed to react with the Cy-chrome-bound streptavidin at
4.degree. C. in the dark for 30 minutes. Staining with the
aforementioned antibodies and Cy-chrome-bound streptavidin and
washing of cells were carried out with the use of 0.1%
NaN.sub.3-containing 1% serum albumin. The cells were stained and
then immobilized with 1% paraformaldehyde-containing PBS(-). Assay
was carried out using the Epics Elite ESP as mentioned above. The
results are shown in Table 1. In Table 1, the percentages (%) of
the IFN-.gamma.-producing NKT cells are shown. This experiment was
separately carried out three times, i.e., experiment 1, experiment
2, and experiment 3. TABLE-US-00001 TABLE 1 Percentage (%) of
IFN-.gamma.- producing NKT cells Sample (mean .+-. S.D.) Experiment
1 Control 2.0 .+-. 0.2 GSL-1 3.6 .+-. 0.3 GSL-2 2.6 .+-. 0.3 GSL-6
4.3 .+-. 1.4 GSL-7 14.0 .+-. 1.4 Experiment 2 Control 3.7 .+-. 0.5
GSL-3 4.6 .+-. 0.6 GSL-4 5.2 .+-. 1.0 GSL-5 4.2 .+-. 0.3 Experiment
3 Control 2.5 .+-. 0.5 GSL-8 4.7 .+-. 0.8 GSL-9 5.4 .+-. 1.2 GSL-10
3.8 .+-. 0.4 GSL-11 5.7 .+-. 0.5 GSL-12 3.2 .+-. 0.2 GSL-13 7.3
.+-. 3.0
[0097] As is apparent from Table 1, effects of accelerating
IFN-.gamma. production were observed. GSL-1, 2, 4, 6 to 11, and 13
were found to be more effective, GSL-6 to 9, 11, and 13 were found
to be further effective, and GSL-7 was found to be remarkably
effective.
Example 3
Effects of Dendritic Cell Activation
[0098] Effects of dendritic cell activation were examined. The
C57BL/6 mice (7-week-old, female) were used. Each type of GSL was
dissolved in the same manner as in Example 2. The samples were
administered to mice through the caudal veins (dose: 100 .mu.g). A
cell suspension was prepared from the spleen extracted 12 hours
after the administration. The leukocytes were treated with the
anti-FcyR antibodies (2.4G2) and then treated with the PE-labeled
anti-CD11c antibodies (Nippon Becton Dickinson Co., Ltd.), the
biotin-labeled anti-CD40 antibodies (Nippon Becton Dickinson Co.,
Ltd.), the biotin-labeled anti-CD80 antibodies (Nippon Becton
Dickinson Co., Ltd.), and the biotin-labeled anti-CD86 antibodies
(Nippon Becton Dickinson Co., Ltd.). After the antibodies were
added to the cells, the reaction was allowed to proceed at
4.degree. C. in the dark for 30 minutes. The cells treated with the
biotin-labeled antibodies were allowed to react with
Cy-chrome-bound streptavidin at 4.degree. C. in the dark for 30
minutes. Staining with the antibodies or Cy-chrome-bound
streptavidin and washing of cells were carried out with the use of
0.1% NaN.sub.3-containing 1% serum albumin. The cells were stained
and then immobilized with 1% paraformaldehyde-containing PBS(-).
Assay was carried out using the Epics Elite ESP. The percentages
(%) of the CD40, CD80, and CD86 positive cells in the CD11c
positive cells are shown in Table 2. TABLE-US-00002 TABLE 2 CD40
(%) CD80 (%) CD86 (%) Sample (mean .+-. S.D.) (mean .+-. S.D.)
(mean .+-. S.D.) Control 51.2 .+-. 0.6 72.8 .+-. 1.3 26.3 .+-. 1.8
GSL-1 67.8 .+-. 1.9 81.9 .+-. 1.2 45.5 .+-. 0.9 GSL-2 65.0 .+-. 9.8
76.2 .+-. 1.8 31.5 .+-. 1.7 GSL-3 58.1 .+-. 2.9 79.9 .+-. 1.3 35.4
.+-. 2.7 GSL-4 63.3 .+-. 0.1 80.6 .+-. 2.6 35.0 .+-. 0.2 GSL-5 59.8
.+-. 0.7 77.4 .+-. 1.1 42.7 .+-. 17.7 GSL-6 65.4 .+-. 2.5 81.0 .+-.
0.4 28.7 .+-. 3.0 GSL-7 78.1 .+-. 3.5 85.4 .+-. 1.7 61.3 .+-. 22.5
GSL-8 73.8 .+-. 4.2 83.9 .+-. 1.0 56.6 .+-. 24.6 GSL-9 76.4 .+-.
1.1 84.9 .+-. 1.3 48.7 .+-. 6.1 GSL-10 64.3 .+-. 4.6 78.9 .+-. 0.4
55.6 .+-. 30.4 GSL-11 71.9 .+-. 2.4 80.9 .+-. 1.2 55.4 .+-. 19.8
GSL-12 64.1 .+-. 2.1 81.3 .+-. 2.0 34.2 .+-. 51.2 GSL-13 77.3 .+-.
0.8 85.2 .+-. 1.3 46.5 .+-. 1.7
[0099] As is apparent from Table 2, effects of dendritic cell
activation were observed. GSL-1, 2, 4, and 6 to 13 were found to be
more effective, and GSL-7 to 9, 11, and 13 were found to be
remarkably effective.
Example 4
Induction of IL-12 and Induction of IL-10
[0100] Bone marrow cells of normal mice that had been used in
Example 1 were cultured in the presence of GM-CSF and IL-4 for 8
days to obtain dendritic cells derived from bone marrow cells. Each
type of GSL shown in the table below was dissolved in an
ethanol:dodecane (98:2) solvent at 0.5 mg/ml, 20 .mu.l each thereof
was dispensed into each well of a 96-well plate, the solvent was
vaporized, and the GSL was immobilized on the plate. As the
control, the ethanol:dodecane (98:2) solvent alone was dispensed
and then vaporized. The dendritic cells derived from bone marrow
cells were applied to the plate onto which GSL had been
immobilized, and the culture supernatant was recovered 24 hours
later. The amount of IL-12 p70 and the amount of IL-10 in the
culture supernatant was measured by ELISA using the BD Opt EIA kit.
The results of the measurement of the amount of IL-12 p70 are shown
in Table 3, and those of IL-10 are shown in Table 4. TABLE-US-00003
TABLE 3 Sample IL-12p70 (pg/ml) Control group Lower than the
detection limit GSL-1 79.3 GSL-2 103.4 GSL-4 231.2 GSL-6 296.6
GSL-8 454.0 GSL-10 547.0 GSL-12 868.6
[0101] TABLE-US-00004 TABLE 4 Sample IL-10 (pg/ml) Control group
129.0 GSL-4 147.4 GSL-10 431.1 GSL-12 583.2 GSL-13 155.9
[0102] As is apparent from the tables, induction of IL-12 p70 and
induction of IL-10 were observed. In particular, for induction of
IL-12 p70, GSL-2, 4, 6, 8, 10, and 12 were found to be more
effective, and GSL-6, 8, 10, and 12 were found to be remarkably
effective. For induction of IL-10, GSL-10 and 12 were found to be
remarkably effective.
Example 5
Effects of NK Cell Activation
[0103] The C57BL/6 mice (7-week-old, female) were used. Each type
of GSL was dissolved in the same manner as in Example 2. The
samples were administered to mice through the caudal veins (dose:
100 .mu.g). As the control, a cell suspension was prepared from the
spleen extracted from the control mice to which the solvent alone
had been administered 16 hours after the administration. The spleen
cells were mixed with the .sup.51Cr-labeled YAC-1 cells at a 50:1
ratio, and those cells were then cultured for 4 hours. The amount
of .sup.51Cr in the culture supernatant 4 hours later was measured
using a .gamma.-counter (Wallac). The results are shown in Table 5.
This experiment was separately carried out three times, i.e.,
experiment 1, experiment 2, and experiment 3. TABLE-US-00005 TABLE
5 NK cell activity (%) Sample (mean .+-. S.D.) Experiment 1 Control
26.1 .+-. 5.1 GSL-2 34.1 .+-. 6.2 GSL-3 33.2 .+-. 8.2 GSL-5 35.7
.+-. 4.8 Experiment 2 Control 17.7 .+-. 2.0 GSL-1 30.1 .+-. 3.8
GSL-6 27.3 .+-. 4.5 GSL-7 33.5 .+-. 5.1 Experiment 3 Control 8.3
.+-. 3.6 GSL-8 22.2 .+-. 1.7 GSL-9 14.8 .+-. 2.1 GSL-10 12.5 .+-.
3.0 GSL-12 11.2 .+-. 3.4 GSL-13 14.4 .+-. 3.1
[0104] As is apparent from Table 5, GSL-1, 5, 6 to 9, and 13 were
found to be more effective, and GSL-1, 7, and 8 were found to be
remarkably effective.
Example 6
Antitumor Effects
[0105] The C57BL/6 mice (7-week-old, female) were used. F16
melanoma F10 (F16F10) tumor cells (the Institute for Genetic
Medicine, Hokkaido University) were administered to mice through
the caudal veins (2.times.10.sup.5 cells/mouse). Each type of GSL
was dissolved in the same manner as in Example 2. The dose of GSL
was 100 .mu.g, and administration thereof was carried out
intraperitoneally 1, 5, and 9 days after the administration of
tumor cells. The mice were subjected to dissection 14 days after
the administration of tumor cells, and the number of metastatic
foci in the lung was counted. The results are shown in Table 6.
TABLE-US-00006 TABLE 6 Number of metastatic foci Sample (solvent)
(mean .+-. S.D.) Control 111.2 .+-. 17.5 GSL-1 (P) 89.5 .+-. 15.9
GSL-2 (P) 40.0 .+-. 15.1 GSL-6 (P) 63.2 .+-. 21.8 GSL-1 (P + S)
69.7 .+-. 16.9 GSL-2 (P + S) 55.7 .+-. 19.8 GSL-6 (P + S) 68.7 .+-.
19.9 GSL-7 (P + S) 67.5 .+-. 14.7
[0106] As is apparent from Table 6, all the above GSLs effectively
inhibited the lung metastases. The effects of GSL-2 were
particularly remarkable.
Example 7
Antiallergic Effects
[0107] The mixed antigens of 100 .mu.g of ovalbumin (OVA) and 1.6
mg of aluminum hydroxide gel were administered via subcutaneous
vaccination to the BALB/c mice on days 0 and day 7. OVA (10 .mu.g)
was dissolved in physiological saline. The OVA (10 .mu.g) dissolved
in physiological saline were administered via nasal vaccination to
the mice on days 14, 15, and 16. The mice were dissected 3 days
after the nasal administration thereof (18 days after the initial
administration). Each type of GSL was dissolved in a solution
containing 0.5% pentanediol, 0.5% N-lauroylsarcosine, and 9.8%
sucrose to a final concentration of 5 mg/ml and then diluted in
physiological saline. The GSL solution diluted in physiological
saline was administered intraperitoneally to mice on days 1, 5, 9,
and 13. The total cell count and eosinophilic leukocyte count in
the alveoli washing fluids were determined. The total cell count
was determined by staining the cells with Turk solution (Wako Pure
Chemical Industries, Ltd.). The eosinophilic leukocyte count was
determined by staining the cells with Giemsa stain (Merck). The
anti-OVA antibody titers (IgG, IgG1, and IgG2a) were assayed by
adding the gradually diluted serum to a 96-well plate coated with
OVA. Alkaline phosphatase-labeled anti-mouse IgG antibodies
(Zymed), anti-mouse IgG1 antibodies (ICN), and anti-mouse-IgG2a
antibodies (Zymed) were used as detection antibodies. The color was
developed using paranitrophenyl phosphate as a substrate, the
reaction was terminated with 3N NaOH, and the absorbance at 405 nm
was measured using a microplate reader. The anti-OVA IgE antibody
titer was assayed by adding the gradually diluted serum to a
96-well plate coated with the anti-mouse IgE antibodies (Nippon
Becton Dickinson Co., Ltd.). Subsequently, biotin-labeled OVA was
added to each well, and the color was developed using
peroxidase-labeled streptavidin and SureBlue (Funakoshi) as a
substrate. After the reaction was terminated with 2N
H.sub.2PO.sub.4, the absorbance at 405 nm was measured using a
microplate reader.
[0108] The control group (the BALB/c mice) was subjected to the
experiment at the same time without GSL administration.
[0109] The results of determining leukocyte count are shown in
Table 7, and those of eosinophilic leukocyte count are shown in
Table 8. TABLE-US-00007 TABLE 7 Leukocyte count 1 .times. 10.sup.5
cells/ml (mean .+-. S.D.) Normal mice 32.7 .+-. 9.9 Control 147.0
.+-. 50.1 GSL-1 88.5 .+-. 34.6
[0110] TABLE-US-00008 TABLE 8 Eosinophilic leukocyte count 1
.times. 10.sup.5 cells/ml (mean .+-. S.D.) Normal mice 0 Control
27.7 .+-. 24.1 GSL-1 4.9 .+-. 4.2
[0111] As is apparent from Table 7 and Table 8, the leukocyte count
and the eosinophilic leukocyte count in the alveoli washing fluids
were significantly decreased.
Example 8
Effects of Enhancing Resistance to Infection
[0112] The C57BL/6 mice (7-week-old, female) were used. Each type
of GSL was dissolved in the same manner as with the case of "P" in
Example 2. The solution was administered intraperitoneally to mice
1 hour before infection with Salmonella typhimurium SL7207 aroA
(Stanford University School of Medicine, U.S.A.). The mice were
dissected 3 days after the infection, and the intraperitoneal
viable cell count was determined. The results are shown in Table 9.
TABLE-US-00009 TABLE 9 Intraperitoneal viable cell count .times.
10.sup.6 cfu Sample (mean .+-. S.D.) Control 20.9 .+-. 3.5 GSL-1
12.1 .+-. 8.2 GSL-2 6.7 .+-. 3.6
[0113] In Table 9, "cfu" is an abbreviation for "colony forming
unit." As is apparent from Table 9, administration of GSL enhanced
the resistance to infection.
Example 9
Antiviral Effects
[0114] The C57BL/6 mice (7-week-old, female) were used. Each type
of GSL was dissolved in the same manner as in Example 2. The
solution was administered intravenously to mice. Mouse
cytomegalovirus (strain Smith, 1.times.10.sup.4 pfu; "pfu" is an
abbreviation for "plaque forming unit") was inoculated
intraperitoneally to mice 1 hour after the administration. The mice
were dissected 3 days after the infection, and the viral titers in
the liver and in the spleen were determined. The results are shown
in Table 10. NK cell activity and serum IFN-.gamma. in the spleen
were measured 3 days after the infection. The results are shown in
Table 11 (NK cell activity) and in Table 12 (IFN-.gamma.).
TABLE-US-00010 TABLE 10 Virus in spleen .times. Virus in liver
.times. 10.sup.3pfu 10.sup.3pfu Sample (mean .+-. S.D.) (mean .+-.
S.D.) Control 2.4 .+-. 0.5 63.0 .+-. 19.5 GSL-1 0.2 .+-. 0.1 7.5
.+-. 4.1 GSL-2 0.2 .+-. 0.1 3.3 .+-. 1.9 GSL-6 0.2 .+-. 0.1 2.7
.+-. 1.2 GSL-7 0.2 .+-. 0.1 4.8 .+-. 1.9
[0115] TABLE-US-00011 TABLE 11 NK cell activity Sample (mean .+-.
S.D.) Control 41.4 .+-. 10.7 GSL-1 72.5 .+-. 9.7 GSL-2 54.0 .+-.
14.0 GSL-6 75.2 .+-. 4.8 GSL-7 72.5 .+-. 7.4
[0116] TABLE-US-00012 TABLE 12 Serum IFN-.gamma. (ng/ml) Sample
(mean .+-. S.D.) Control 2.0 .+-. 0.5 GSL-1 3.0 .+-. 0.7 GSL-2 4.5
.+-. 1.1 GSL-6 2.6 .+-. 0.7 GSL-7 2.5 .+-. 0.3
[0117] As shown in Table 10, pre-administration of GSL resulted in
the enhancement of resistance to infection. As shown in Table 11,
NK cell activity was significantly enhanced, and the IFN-.gamma.
level was enhanced, compared with the control group. The effects of
GSL-6 were found to be particularly significant.
Example 10
Acceleration of IL-6 Production and Acceleration of NO
Production
[0118] Thioglycolate broth (4%, 3 ml) was administered
intraperitoneally to the normal mice that were employed in example
1, and the peritoneal exudate cells were sampled therefrom 4 days
later and then subjected to the experiment. The macrophage cell
line RAW 264 (RIKEN) was also subjected to the experiment. Each
type of GSL was dissolved in an ethanol:dodecane (98:2) solvent at
0.5 mg/ml, 20 .mu.l each thereof was dispensed into each well of a
96-well plate, the solvent was vaporized, and the GSL was
immobilized onto the plate. The macrophage was added to the plate
onto which GSL had been immobilized, and the culture supernatant
was recovered 24 hours thereafter. The amount of IL-6 in the
supernatant was measured via ELISA and the amount of nitrogen
monoxide (NO) was measured using the Griess reagent. The results of
measuring IL-6 are shown in Table 13, and those of measuring
nitrogen monoxide are shown in Table 14. TABLE-US-00013 TABLE 13
IL-6 (pg/ml) Sample (mean .+-. S.D.) Control 3491.0 .+-. 492.1
GSL-1 3921.7 .+-. 665.1 GSL-4 3453.0 .+-. 252.5 GSL-5 4742.0 .+-.
525.2 GSL-6 5389.7 .+-. 1003.7 GSL-7 6948.0 .+-. 457.1 GSL-8 6264.0
.+-. 1075.0 GSL-9 6711.3 .+-. 485.1 GSL-10 7474.0 .+-. 920.3 GSL-11
10046.0 .+-. 340.0 GSL-12 7303.3 .+-. 640.2 GSL-13 7619.3 .+-.
774.3
[0119] TABLE-US-00014 TABLE 14 NO (.mu.M) Sample (mean .+-. S.D.)
Control group 0.7 .+-. 0.2 GSL-2 6.8 .+-. 0.7 GSL-3 3.5 .+-. 0.2
GSL-4 3.8 .+-. 0.5 GSL-5 5.2 .+-. 0.5 GSL-6 4.4 .+-. 0.8 GSL-7 3.9
.+-. 0.3 GSL-8 4.2 .+-. 1.1 GSL-9 9.0 .+-. 0.5 GSL-10 8.4 .+-. 0.8
GSL-11 11.3 .+-. 1.0 GSL-12 7.7 .+-. 0.5 GSL-13 7.1 .+-. 0.1
[0120] As is apparent from Table 13, effects of IL-6 induction were
observed. GSL-5 to 13 were found to be more effective, GSL-6 to 13
were found to be further effective, and GSL-7 to 13 were found to
be remarkably effective.
[0121] As is apparent from Table 14, effects of accelerating NO
production were observed. GSL-2, 5, and 9 to 13 were found to be
more effective, and GSL-11 was further effective.
Example 11
[0122] The toxicities of the GSL compositions according to the
present invention were examined. The C57BL/6 mice (7-week-old,
female) were used. Galactosamine (20 mg) was administered
intraperitoneally to mice, and immediately thereafter,
lipopolysaccharides (hereafter the same may be abbreviated as
"LPS," derived from Salmonella abortus equi, the Max-Planck
Institute for Immunobiology, Freiburg, Germany) (solvent:
physiological saline) or GSL shown in. Table 15 (solvent: "P+S")
were administered to mice via the caudal veins in amounts shown in
Table 15. Whether or not the mice survived was examined 24 hours
after the administration. The results are shown in Table 15.
TABLE-US-00015 TABLE 15 Number of mice died/Number LPS or GSL
Amount administered of mice tested GSL-1 100 .mu.g 0/3 GSL-2 100
.mu.g 0/3 GSL-3 100 .mu.g 0/3 GSL-4 100 .mu.g 0/3 GSL-5 100 .mu.g
0/3 GSL-6 100 .mu.g 0/3 GSL-7 100 .mu.g 0/3 GSL-8 100 .mu.g 0/3
GSL-9 100 .mu.g 0/3 GSL-10 100 .mu.g 0/3 GSL-11 100 .mu.g 0/3
GSL-12 100 .mu.g 0/3 GSL-13 100 .mu.g 0/3 LPS 100 ng 3/3 LPS 10 ng
3/3
[0123] When LPS was administered, all mice died even with the
administration of 10 ng thereof. On the contrary, all mice survived
with the administration of GSL in amounts of as much as 100 .mu.g.
Thus, the toxicities of the compositions according to the present
invention were found to be very low.
Example 12
[0124] Whether or not endotoxin tolerance was induced was examined.
The C57BL/6 mice (7-week-old, female) were used. LPS (solvent:
physiological saline) or GSL was administered to mice via the
caudal veins in amounts shown in Table 16. Galactosamine (20 mg)
and LPS (100 ng, the solvent: physiological saline) were
administered intraperitoneally to mice 24 hours after the
administration, and whether or not the mice survived was examined.
The results are shown in Table 16. TABLE-US-00016 TABLE 16 Number
of mice died/Number LPS or GSL Amount administered of mice tested
GSL-1 100 .mu.g 3/3 GSL-2 100 .mu.g 3/3 GSL-6 100 .mu.g 3/3 GSL-7
100 .mu.g 3/3 LPS 100 ng 0/3
[0125] As is apparent from Table 16, endotoxin tolerance was not
induced with the administration of 100 .mu.g of GSL-1, GSL-2,
GSL-6, or GSL-7, and all mice died. On the contrary, endotoxin
tolerance was induced with the administration of 100 ng of LPS.
[0126] It was thus found that the compositions of the present
invention are more effective for NKT cell activation. Also, a
composition for accelerating IL-4 production, a composition for
accelerating IFN-.gamma. production, a composition for dendritic
cell activation, a composition for accelerating IL-12 production, a
composition for accelerating IL-10 production, a composition for NK
cell activation, an antitumor composition, an antiallergic
composition, a composition for enhancing resistance to infection,
an antiviral composition, a composition for accelerating IL-6
production, and a composition for accelerating NO production were
obtained. The compositions of the present invention are
particularly useful since they have such functions at once.
[0127] Further, the compositions of the present invention are
useful since they have few side effects, i.e. the toxicities
thereof are low and endotoxin tolerance is not induced.
[0128] The present disclosure relates to the subject matter
contained in Japanese Patent Application No. 043481/2004 filed on
Feb. 19, 2004 and PCT/JP2005/003234 filed on Feb. 21, 2005, which
are expressly incorporated herein by reference in theirs
entirety.
[0129] The foregoing description of preferred embodiments of the
invention has been presented for purposes of illustration and
description, and is not intended to be exhaustive or to limit the
invention to the precise form disclosed. The description was
selected to best explain the principles of the invention and their
practical application to enable others skilled in the art to best
utilize the invention in various embodiments and various
modifications as are suited to the particular use contemplated. It
is intended that the scope of the invention not be limited by the
specification, but be defined claims set forth below.
* * * * *